SUMO proteins are a family of ubiquitin-related proteins that become covalently linked to other cellular proteins. While budding yeast has a single SUMO, called Smt3p, there are three commonly expressed mammalian SUMO paralogues, called SUMO-1, -2 and -3. SUMO-2 and -3 are 96% identical, while SUMO-1 is roughly 45% identical to either SUMO-2 or -3. In this report, SUMO-2 and -3 will be collectively called SUMO-2/3 under circumstances where they cannot be distinguished from each other. The individual functions of vertebrate SUMO paralogues are poorly understood: SUMO-1 is less abundant than SUMO-2/3, and it is conjugated to a distinct spectrum of targets. SUMO-1 also has different in vivo dynamics and responses to physiological stress such as heat shock. Unlike SUMO-2/3, SUMO-1 is concentrated at nuclear pore complexes (NPCs), the primary conduit of nucleocytoplasmic trafficking, reflecting the fact that it is the preferred conjugation partner of RanGAP1. RanGAP1 is the GTPase activator for Ran, a small GTPase that controls nuclear transport. SUMO-1 conjugation of RanGAP1 promotes its stable association to the NPCs through binding to a large nucleoporin, Nup358/RanBP2.[unreadable] Human SUMO proteins have been implicated in a variety of cell functions, including nuclear trafficking, chromosome segregation, chromatin organization, transcription and RNA metabolism. The conjugation pathway for SUMO proteins is similar to the ubiquitin conjugation pathway: SUMO proteins are processed by Ubiquitin like proteases/Sentrin specific proteases (Ulps/SENPs) to reveal a di-glycine motif at their C-termini. After processing, SUMO proteins undergo ATP-dependent formation of a thioester bond to their activating (E1) enzyme, Aos1/Uba2. The activated SUMO proteins are transferred to form a thioester linkage with their conjugating (E2) enzyme, Ubc9. Finally, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). The linkage of SUMO proteins to their substrates can be severed by Ulps/SENPs, so it is likely that SUMO modification is highly dynamic in vivo. Ulp/SENPs play an important role in determination of the spectrum of conjugated species because they directly regulate the production of free, conjugatable SUMO proteins and the half-life of conjugated species. There are six members of the Ulp/SENP family in mammals and five in amphibians (Xenopus laevis). We are systematically evaluating the physiological roles and regulation of these enzymes.[unreadable] We have found that two Ulp/SENP family members, SENP3 and SENP5, localize within the granular component of the nucleolus, a sub-nucleolar compartment that contains B23/Nucleophosmin. B23/Nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis, and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/Nucleophosmin binds SENP3 and SENP5 in Xenopus egg extracts, and that B23/Nucleophosmin promotes the stability of SENP3 and SENP5 in mammalian tissue culture cells. After either co-depletion of SENP3 and SENP5 or depletion of B23/Nucleophosmin, we observe accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/Nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/Nucleophosmin function in vivo. We are currently working to understand how SUMOylation contributes toward ribosome biogenesis through examination of SENP3 and SENP5 regulation, as well as through the identification of ribosomal SUMOylation targets. [unreadable] We had previously shown that SENP6 (also called SUSP1) localizes within the nucleoplasm, where it plays a specialized role in dismantling highly conjugated SUMO-2 and -3 species. This function is similar to the chain-editing activity of SENP6s closest relative in budding yeast, Ulp2. We have examined the role of SENP6 in chromosome segregation, a process that requires Ulp2p in yeast. We find that siRNA-mediated knockdown of SENP6 in HeLa cells leads to chromosome misalignment. This phenotype is accompanied by defects in spindle assembly and mitotic progression. To understand the underlying molecular defect, we have systematically evaluated the behavior of kinetochore-associated proteins, particularly those found to be SUMOylated in other contexts. We find both microtubule-dependent and microtubule-independent changes in kinetochore composition, and are currently working to establish whether these alterations can be directly linked to the modification of individual kinetochore proteins.